This invention relates to the filed of polynucleotide amplification. More particularly, the invention provides methods, compositions and kits for amplifying (i.e., making multiple copies) target polynucleotide sequences which involve transcription, and employ either an RNA/DNA composite primer and/or a target switch polynucleotide.
The development of methods for nucleic acid amplification and detection of amplification products have advanced the detection, identification, quantification and sequence analyses of nucleic acids in recent years. Use of these methods has contributed to rapid advances in the areas of genomics, cell biology, molecular medicine, genetics and the like.
Nucleic acid analysis is widely used for detection and identification of pathogens, detection of gene alterations leading to defined phenotypes, diagnosis of genetic diseases or susceptibility to such disease, assessment of gene expression in development, in disease and in response to defined stimuli, as well as in various genome projects. Other applications of nucleic acid amplification methods include the detection of rare cells, detection of pathogens and the detection of altered gene expression in malignancy, and the like. Nucleic acid amplification is potentially useful for both qualitative analysis such as the detection of the presence of defined nucleic acid sequences, and quantification of defined gene sequences. The latter is useful for assessing and determining the amount of pathogenic sequences in a sample as well as for the determination of gene multiplication or deletion, as often found in cell transformation from normal to malignant type.
Although detection of the presence of a defined nucleic acid sequence, and its sequence analysis, can be carried out by direct probe hybridization to target nucleic acid sequences, the method generally lacks sensitivity when amounts of the target nucleic acid sequence present in the test sample are low. One solution to this obstacle was the development of methods for generation of multiple copies of the defined nucleic acid sequence to make them more accessible to further analysis. Methods for generating multiple copies of a specific nucleic acid sequence in a sample are generally defined as target amplification methods. Other methods for increasing the sensitivity of detection of hybridization analysis are based on the generation of multiple products from the hybridized probe(s), for example, cleaving the hybridized probe to form multiple products or ligating adjacent probes to form a unique hybridization-dependent product. Similarly, increased sensitivity of hybridization reaction is achieved by methods for amplifying signals generated by the hybridization event, such as methods based on hybridization of branched DNA probes.
Various target nucleic acid amplification methods have been described in recent years. Target nucleic acid amplification is carried out through multiple cycles of incubations at various temperatures (thermal cycling) or alternatively, carried out by an isothermal process. The discovery of thermostable nucleic acid modifying enzymes has also contributed to rapid advances in nucleic acid amplification technology. Thermostable nucleic acid modifying enzymes, such as DNA and RNA polymerases, ligases, nucleases and the like, are used both in methods dependent on thermal cycling and isothermal amplification methods. For example, a method for xe2x80x9chomogeneous isothermal amplification and detection of nucleic acids using a template switch oligonucleotidexe2x80x9d is described in WO 00/70095 A2 (Liu, et al.).
The most commonly used target amplification method is the polymerase chain reaction (PCR) which is based on multiple cycles of denaturation, hybridization of two oligonucleotide primers, one to each strand of a double stranded target, and primer extension by a nucleotide polymerase to produce multiple double stranded copies of the target sequence. Many variations of PCR have been described, and the method is being used for amplification of DNA or RNA nucleic acid sequences, sequence determination, mutation analysis and others. (see PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J. and White, T., eds.) Academic Press (1990); Mullis et al., Methods in Enzymology, 155:335-350 (1987)). Thermocycling-based methods that employ a single primer are also described. Other methods that depend on thermal cycling include the ligase chain reaction (LCR) and the related repair chain reaction (RCR).
Isothermal nucleic acid amplification methods based on strand displacement, are described. See, for e.g., Fraiser et al. in U.S. Pat. No. 5,648,211; Cleuziat et al. in U.S. Pat. No. 5,824,517; and Walker et al. Proc. Natl. Acad. Sci. U.S.A. 89:392-396 (1992). Other isothermal target amplification methods are the transcription-based amplification methods, in which an RNA polymerase promoter sequence is incorporated into primer extension products at an early stage of the amplification (WO 98/01050), and target sequence, or target complementary sequence, is further amplified by transcription and digestion of the RNA strand in a DNA/RNA hybrid intermediate product. See, for example, U.S. Pat. Nos. 5,169,766 and 4,786,600. Target nucleic acid amplification may be carried out through multiple cycles of incubations at various temperatures, i.e. thermal cycling, or at one temperature (an isothermal process). These methods include transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), and variations thereof. See, for example, Guatelli et al. Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878 (1990); U.S. Pat. No. 5,766,849 (TMA); and U.S. Pat. No. 5,654,142 (NASBA). Other amplifications methods use template switching oligonucleotides (TSOs) and blocking oligonucleotides. For example, a template switch amplification method in which chimeric DNA primers are utilized is disclosed in U.S. Pat. No. 5,679,512 and by Patel et al. (Proc. Nail. Acad. Sci. U.S.A. 93:2969-2974 (1996)), and a method that uses blocking oligonucleotides is disclosed by Laney et al. in U.S. Pat. No. 5,679,512.
Since isothermal target amplification methods do not require a thermocycler, they are easier to adapt to common instrumentation platforms. However, previously known isothermal target amplification methods have severe drawbacks. Amplification according to the strand displacement amplification (SDA) process requires the presence of sites for defined restriction enzymes, thus limiting its applicability. Existing transcription-based amplification methods, such as the NASBA and TMA, on the other hand, are limited by the need for incorporation of the RNA polymerase promoter sequence into the amplification product by means of a primer, a process prone to causing non-specific amplification. Moreover, the mechanism of amplification of a DNA target by these transcription-based amplification methods is not well established.
The completion of sequencing of a number of genomes, and the sequencing of the human genome in particular, has tremendous implications for advances in molecular and cell biology in general and molecular medicine in particular. Development of methods for isothermal gene sequencing will greatly enhance the application of this information in various testing facilities, as isothermal sequencing markedly simplifies the process of genetic identification compared to current methods that require thermocycling.
The methods of the present invention provide for isothermal, high efficiency nucleic acid sequence amplification and methods that use these amplification methods and products, such as in sequence determination.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.
The invention provides methods, compositions and kits for amplifying polynucleotide sequences. The methods generally comprise use of a primer (which in some aspects of the invention is an RNA/DNA composite primer), optionally a termination sequence, and a propromoter oligonucleotide sequence.
Accordingly, in one aspect the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising the steps of: (a) hybridizing a single stranded target polynucleotide comprising the nucleic acid sequence of interest with a first primer, wherein said first primer is a composite primer comprising an RNA portion and a 3xe2x80x2 DNA portion; (b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the target polynucleotide 5xe2x80x2 with respect to hybridization of the first primer to the target polynucleotide; (c) extending the first primer with a DNA-dependent DNA polymerase to generate a complex comprising a first primer extension product and target polynucleotide; (d) cleaving the RNA portion from the composite primer in the complex of first primer extension product and target polynucleotide with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer (which is generally and preferably the same as the first primer) can hybridize to the target polynucleotide and repeat primer extension by strand displacement to produce displaced primer extension product; (e) hybridizing a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase (these conditions generally but not necessarily include extension of the 3xe2x80x2 end of the primer extension product to generate a double stranded promoter region), such that an RNA transcript comprising sequences complementary to the displaced primer extension products is produced; (f) hybridizing a second primer to the RNA transcript of step (e); (g) extending the second primer with RNA-dependent DNA polymerase to generate a complex comprising a second primer extension product and the RNA transcript; (h) cleaving RNA in the complex of step (g) with an enzyme that cleaves RNA in an RNA/DNA hybrid; (i) hybridizing the single stranded second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase (these conditions generally but not necessarily include extension of the 3xe2x80x2 end of the primer extension product to generate a double stranded promoter region), such that an RNA transcript comprising the sequence of interest is produced; whereby multiple copies of the nucleic acid sequence of interest are produced.
In another aspect the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising (a) combining: the complex comprising a first primer extension product and target polynucleotide as described above; a composite primer (which is generally and preferably the same as the first primer) that is hybridizable to the target polynucleotide, wherein the composite primer is a primer that comprises an RNA portion and a 3xe2x80x2 DNA portion; an enzyme that cleaves RNA from an RNA/DNA hybrid; a propromoter polynucleotide comprising a propromoter and a region which hybridizes to displaced composite primer extension product; an RNA polymerase; a second primer that is hybridizable to a sense RNA transcript comprising the sequence of interest; an RNA-dependent DNA polymerase; and a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and (b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, displacement of the first primer extension product from the complex comprising a first primer extension product and target polynucleotide when its RNA is cleaved and a composite primer binds to the target polynucleotide in the complex, hybridization of a propromoter polynucleotide to a first primer extension product to form a complex comprising a first primer extension product and a propromoter polynucleotide, hybridization of a propromoter polynucleotide to a second primer extension product to form a complex comprising a second primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.
In another aspect the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising: (a) combining: the displaced primer extension product generated as described above; a propromoter polynucleotide comprising a propromoter and a region which hybridizes to displaced first primer extension product; an RNA polymerase; a second primer that is hybridizable to a sense RNA transcript comprising the sequence of interest; an RNA-dependent DNA polymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; and a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and (b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, hybridization of a propromoter polynucleotide to a first primer extension product to form a complex comprising a first primer extension product and a propromoter polynucleotide, hybridization of a propromoter polynucleotide to a second primer extension product to form a complex comprising a second primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.
In another aspect the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising: (a) combining: an RNA transcript comprising sequences complementary to the displaced primer extension products as described above; a second primer that is hybridizable to a sense RNA transcript comprising the sequence of interest; an RNA-dependent DNA polymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and an RNA polymerase; and (b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, hybridization of a propromoter polynucleotide to a primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.
In yet another aspect the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising: (a) combining: a first primer, wherein the first primer is a composite primer that is hybridizable to a target polynucleotide, and wherein the composite primer comprises an RNA portion and a 3xe2x80x2 DNA portion; optionally a polynucleotide comprising a termination polynucleotide sequence that is hybridizable to a region of the target polynucleotide which is 5xe2x80x2 with respect to hybridization of the composite primer to the target polynucleotide; a DNA-dependent DNA polymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a first primer extension product; an RNA polymerase; a second primer that is hybridizable to a sense RNA transcript comprising the sequence of interest; an RNA-dependent DNA polymerase; and a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and (b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, displacement of a first primer extension product from a complex comprising a first primer extension product and target polynucleotide when its RNA is cleaved and a composite primer binds to the target polynucleotide in the complex, hybridization of a propromoter polynucleotide to a primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.
In another aspect, the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising: (a) hybridizing a second primer to an RNA transcript, said RNA transcript produced by a process comprising (i) hybridizing a single stranded target polynucleotide comprising the nucleic acid sequence of interest with a first primer, wherein said first primer is a composite primer comprising an RNA portion and a 3xe2x80x2 DNA portion; (ii) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the target polynucleotide 5xe2x80x2 with respect to hybridization of the first primer to the target polynucleotide; (iii) extending the first primer with a DNA-dependent DNA polymerase to generate a complex comprising a first primer extension product and target polynucleotide; (iv) cleaving the RNA portion from the composite primer in the complex of first primer extension product and target polynucleotide with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer (which is generally and preferably the same as the first primer) can hybridize to the target polynucleotide and repeat primer extension by strand displacement to produce displaced primer extension product; (v) hybridizing a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase (these conditions generally but not necessarily include extension of the 3xe2x80x2 end of the primer extension product to generate a double stranded promoter region), such that an RNA transcript comprising sequences complementary to the displaced primer extension product is produced; and (b) extending the second primer with RNA-dependent DNA polymerase to generate a complex comprising a second primer extension product and the RNA transcript; (c) cleaving RNA in the complex of step (b) with an enzyme that cleaves RNA in an RNA/DNA hybrid; (d) hybridizing the single stranded second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase (these conditions generally but not necessarily include extension of the 3xe2x80x2 of the primer extension product to generate a double stranded promoter region), such that an RNA transcript comprising the sequence of interest is produced; whereby multiple copies of the nucleic acid sequence of interest are produced.
Various embodiments of the composite primer used in the composite primer-based methods of the invention are described herein. For example, in some embodiments, the RNA portion of the composite primer is 5xe2x80x2 with respect to the 3xe2x80x2 DNA portion. In still other embodiments, the 5xe2x80x2 RNA portion is adjacent to the 3xe2x80x2 DNA portion. For the composite primer-based methods described herein, one or more composite primers can be used. In preferred embodiments of the composite primer-based methods, the first and second primers are different (for example, the first primer is a composite primer and the second primer is not a composite primer). In preferred embodiments wherein the first and second primers are different, the primers are hybridizable to similar, preferably identical, sequences. In still other embodiments of the composite primer-based methods, the first and second primers are hybridizable to different sequences.
Various exemplary embodiments of polynucleotides comprising a termination sequence are also described herein. In some embodiments, the polynucleotide comprising a termination sequence is a template switch oligonucleotide (TSO), which may (but not necessarily) contain one or more modifications to enhance binding to template. Accordingly, in some embodiments, the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification. Examples of suitable modifications are provided herein. In some embodiments, the polynucleotide comprising a termination sequence is a blocking sequence, which, like the TSO, may contain one or more modifications to enhance binding to template. Accordingly, in some embodiments, the blocker sequence comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the blocker binds more tightly to the region as compared to a blocker without the modification. Examples of suitable modifications are provided herein.
The enzymes which may be used in the methods and compositions are described herein. For example, the enzyme that cleaves RNA may be an RNaseH.
In some aspects, a TSO provides propromoter function and also comprises a region (which may or may not be adjacent to the promoter) which hybridizes to the displaced primer extension product. In other embodiments, the polynucleotide comprising the propromoter comprises a region at the 3xe2x80x2 end which hybridizes to the displaced primer extension product, whereby DNA polymerase extension of displaced extension product produces a double stranded promoter from which transcription occurs. In some embodiments, the polynucleotide comprising the propromoter is a propromoter template oligonucleotide (PTO). In some embodiments, the polynucleotide comprising the propromoter is a polynucleotide comprising: (a) a termination sequence that does not effect template switch under conditions wherein the termination sequence is hybridizable to a target polynucleotide; (b) a propromoter sequence, wherein the propromoter sequence is not hybridizable to a target polynucleotide under conditions wherein the termination sequence is hybridizable to the target polynucleotide; and (c) a sequence which is hybridizable to a complementary sequence of said target polynucleotide.
In another aspect the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising: (a) hybridizing a single stranded target polynucleotide comprising the sequence of interest with a first primer; (b) hybridizing a propromoter template switch oligonucleotide (TSO) comprising a propromoter sequence and a region that is hybridizable to a region of the target polynucleotide which is 5xe2x80x2 with respect to hybridization of the first primer to the target polynucleotide; (c) extending the first primer with DNA polymerase such that a first primer extension product comprising a sequence complementary to the propromoter sequence of the propromoter TSO is produced, whereby a complex of first primer extension product, target polynucleotide and propromoter TSO is generated, wherein said complex comprises a double stranded promoter region; (d) transcribing from the double stranded promoter region with a DNA-dependent RNA polymerase to generate a sense RNA transcript; (e) hybridizing a second primer (which is generally and preferably the same as the first primer) to the sense RNA transcript of step (d); (f) extending the second primer with RNA-dependent DNA polymerase to generate a complex comprising a second primer extension product and an RNA transcript; (g) cleaving RNA in the complex of step (f) with an enzyme that cleaves RNA in an RNA/DNA hybrid; (h) hybridizing a single stranded second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that sense RNA transcripts comprising the sequence of interest are produced; whereby multiple copies of the nucleic acid sequence of interest are produced.
In another aspect the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising: (a) combining: the complex described above of a first primer extension product, target polynucleotide and propromoter TSO, wherein said complex comprises a double stranded promoter region; an RNA polymerase; a second primer (which is generally and preferably the same as the first primer) that is hybridizable to a sense RNA transcript comprising the sequence of interest; an RNA-dependent DNA polymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; and a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and (b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, hybridization of a propromoter polynucleotide to a primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.
In another aspect the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising: (a) combining: a sense RNA transcript generated as described above; a second primer (which is generally and preferably the same as the first primer) that is hybridizable to the RNA transcript; an RNA-dependent DNA polymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and an RNA polymerase; and (b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, hybridization of a propromoter polynucleotide to a primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid of interest are generated.
In another aspect the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising: (a) combining: a target polynucleotide; a first primer which is hybridizable to the target polynucleotide; a propromoter template switch oligonucleotide comprising a propromoter sequence and a region that is hybridizable to a region of the target polynucleotide which is 5xe2x80x2 with respect to hybridization of the first primer to the target polynucleotide; optionally a DNA-dependent DNA polymerase; an RNA polymerase; a second primer (which is generally and preferably the same as the first primer) that is hybridizable to a sense RNA transcript comprising the sequence of interest; an RNA-dependent DNA polymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; and a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and (b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, hybridization of a propromoter polynucleotide to a primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.
In another aspect, the invention provides methods for generating multiple copies of a nucleic acid sequence of interest, comprising: (a) hybridizing a second primer to an RNA transcript, said RNA transcript produced by a process comprising (i) hybridizing a single stranded target polynucleotide comprising the sequence of interest with a first primer (which is generally and preferably the same as the second primer); (ii) hybridizing a propromoter template switch oligonucleotide (TSO) comprising a propromoter sequence and a region that is hybridizable to a region of the target polynucleotide which is 5xe2x80x2 with respect to hybridization of the first primer to the target polynucleotide; (iii) extending the first primer with DNA polymerase such that a first primer extension product comprising a sequence complementary to the propromoter sequence of the propromoter TSO is produced, whereby a complex of first primer extension product, target polynucleotide and propromoter TSO is generated, wherein said complex comprises a double stranded promoter region; (iv) transcribing from the double stranded promoter region with a DNA-dependent RNA polymerase to generate a sense RNA transcript; (b) extending the second primer with RNA-dependent DNA polymerase to generate a complex comprising a second primer extension product and an RNA transcript; (c) cleaving RNA in the complex of step (b) with an enzyme that cleaves RNA in an RNA/DNA hybrid; (d) hybridizing a single stranded second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that sense RNA transcripts comprising the sequence of interest are produced; whereby multiple copies of the nucleic acid sequence of interest are produced.
In preferred embodiments of the propromoter TSO-based methods, the first primer and the second primer are the same. In other embodiments of the propromoter TSO-based methods, the first and second primers are different. In certain embodiments wherein the first and second primers are different, the primers are hybridizable to similar, preferably identical, sequences. In still other embodiments of the propromoter TSO-based methods, the first and second primers are hybridizable to different sequences.
The methods are applicable to amplifying any DNA or RNA target, including, for example, genomic DNA or RNA and cDNA. One or more steps may be combined and/or performed sequentially (often in any order, as long as the requisite product(s) are formed).
The invention also provides methods which employ (usually, analyze) the products of the amplification methods of the invention, such as sequencing and detection of sequence alteration(s). A method of sequencing according to the invention comprises: (a) amplifying a target polynucleotide by the methods of the invention in the presence of a mixture of rNTPs and rNTP analogs such that transcription is terminated upon incorporation of an rNTP analog; and (b) analyzing the amplification products to determine sequence.
Another method of sequencing according to the invention comprises: (a) amplifying a target polynucleotide by the methods of the invention wherein RNA transcripts generated from a first primer extension product are amplified in the presence of a mixture of rNTPs and rNTP analogs such that transcription is terminated upon incorporation of an rNTP analog; and (b) analyzing the amplification products to determine sequence. Another method of sequence analysis according to the invention comprises: (a) amplifying a target polynucleotide by the methods of the invention; and (b) analyzing the amplification products for single stranded conformation, wherein a difference in conformation as compared to a reference single stranded polynucleotide indicates a mutation in the target polynucleotide sequence.
The invention provides methods of characterizing a sequence of interest in a target polynucleotide comprising (i) amplifying a target polynucleotide sequence containing the sequence of interest by the methods of the invention, wherein the sequence of the RNA portion of the composite primer is known, and (ii) comparing the amplification products if any from step (i) with the amount of amplification products from a reference template wherein (1) production of detectably fewer amplification products from the template as compared to the amount of amplification products from the reference template which comprises a region complementary to the RNA portion of the composite primer indicates that the target polynucleotide does not comprise a sequence complementary to the RNA portion of the composite primer and is a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer; or (2) production of detectably more amplification products from the template as compared to the amount of amplification products from the reference template which does not comprise a region which is complementary to the RNA portion of the composite primer indicates that the target polynucleotide comprises a sequence complementary to the RNA portion of the composite primer and is not a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer.
In the methods of the invention the sequence of the RNA portion of the composite primer may comprise a sequence complementary to the sequence of interest which may be a wild type sequence, a mutant sequence, or an allelic variant sequence.
The methods of the invention are useful for detection and quantification of a target nucleic acid sequence.
For example, in one aspect, the invention provides methods of sequencing a sequence of interest comprising sequencing a nucleic acid amplification product, wherein said nucleic acid amplification product is generated by an amplification method described herein. Sequencing of the amplification products reveals the sequence of the sequence of interest.
In yet another example, the invention provides methods of detecting presence of a nucleic acid sequence of interest in a sample, said method comprising detecting presence of the sequence of interest in a nucleic acid amplification product generated by an amplification method described herein. Detection of the sequence of interest in the amplification products is indicative of presence of the sequence of interest in a sample.
In various embodiments of methods of the invention, a sequence of interest comprises a mutation. In some embodiments wherein a sequence of interest comprises a mutation, the mutation is a single nucleotide polymorphism. In some embodiments, detecting of a sequence of interest comprises hybridizing an amplification product comprising the sequence of interest with a nucleic acid probe that is hybridizable to said nucleic acid sequence of interest. In other embodiments, detecting of a sequence of interest comprises hybridizing an amplification product comprising the complement of the sequence of interest with a nucleic acid probe that is hybridizable to said complement. In some embodiments, a nucleic acid probe comprises DNA. In other embodiments, a nucleic acid probe comprises RNA. In some embodiments, a probe is provided as a microarray. In some embodiments, a microarray comprises a probe immobilized on a substrate fabricated from a material selected from the group consisting of paper, glass, plastic, polypropylene, nylon, polyacrylamide, nitrocellulose, silicon, and optical fiber. In some embodiments, a sequence of interest or its complement is detected by conducting limited primer extension, wherein the limited primer extension comprises extending a primer hybridized to an amplification product such that a primer extension product is generated that has a characteristic that indicates presence or absence of the sequence of interest.
Thus, the methods of the invention are useful for detection and quantification of a target nucleic acid sequence. The single-stranded nucleic acid amplification products of the invention can also be used for the detection and quantification of a target nucleic acid sequence by hybridization to probes, such as immobilized probes. The invention also provides methods of producing a microarray, comprising (a) amplifying a target polynucleotide by the methods of the invention; and (b) immobilizing the amplification products on a substrate to fabricate a microarray comprising the amplification products.
One aspect of the invention provides methods of determining gene expression profile in a sample comprising: (a) amplifying at least two sequences of interest in the sample by the methods of the invention; and (b) determining amount of amplification products of each sequence of interest, wherein each said amount is indicative of amount of each sequence of interest in the sample, whereby the gene expression profile in the sample is determined. In one embodiment each target polynucleotide is a cDNA. In another embodiment each target polynucleotide is RNA.
The invention also provides compositions, kits, complexes, reaction mixtures and systems comprising various components (and various combinations of the components) used in the amplification methods described herein. One aspect of the invention provides a system for amplifying a sequence of interest, comprising: (a) a first primer which is a composite primer; (b) a second primer; (c) a DNA-dependent DNA polymerase; (d) an RNA-dependent DNA polymerase; (e) a propromoter polynucleotide; (f) an RNA polymerase; and (g) an enzyme that cleaves RNA from an RNA/DNA hybrid; and optionally, (h) a polynucleotide comprising a termination polynucleotide sequence. Another aspect of the invention provides a system for amplifying a sequence of interest, comprising: (a) a propromoter TSO; (b) a first primer; (c) optionally a DNA-dependent DNA polymerase; (d) an RNA-dependent DNA polymerase; (e) an enzyme that cleaves RNA from an RNA/DNA hybrid; (f) an RNA polymerase; and optionally (g) a second primer.
In another aspect, the invention provides reaction mixtures (or compositions comprising reaction mixtures) which contain various combinations of components described herein. In another aspect, the invention provides kits for conducting the methods described herein. These kits, in suitable packaging and generally (but not necessarily) containing suitable instructions, contain one or more components used in the amplification methods. For example, the invention provides kits that comprise a composite primer (for example, comprising a 3xe2x80x2 DNA portion and an RNA portion, which may be 5xe2x80x2 and may further be adjacent to the 3xe2x80x2 DNA portion) that is hybridizable to a target polynucleotide, a propromoter polynucleotide, and instructions for amplifying the target polynucleotide according to any composite primer-based method described herein. The composite primer and propromoter polynucleotide in the kits can be any described herein. The kits can contain further components, such as any of (a) a polynucleotide comprising a termination polynucleotide sequence; (b) any of the enzymes described herein, such as an enzyme which cleaves RNA from an RNA/DNA hybrid (for example, RNaseH); (c) a propromoter TSO; and (d) a second primer. In another example, the invention provides kits comprising a propromoter TSO and a first primer (which may or may not be a composite primer), wherein both are hybridizable to a target polynucleotide, and instructions for amplifying the target polynucleotide according to any propromoter TSO-based method described herein. The kits can contain further components, such as any of (a) any of the enzymes described herein, such as an enzyme which cleaves RNA from an RNA/DNA hybrid (for example, RNaseH); (b) a polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and (c) optionally a second primer.